Antigen profiles for the quantitative assessment of eosinophils in mouse tissues by flow cytometry

Eosinophil Biology Section, Laboratory of Allergic Diseases, NIAID, NIH Bethesda, MD, United States.
Journal of immunological methods (Impact Factor: 1.82). 06/2011; 369(1-2):91-7. DOI: 10.1016/j.jim.2011.04.009
Source: PubMed


Much of our current understanding of eosinophil-associated pathologies has developed from the use of mouse models. While mouse eosinophils can be readily detected by flow cytometric methods, most studies do not document the efficiency of this process compared to direct counting of stained cells. Our intent was to address this knowledge gap by identifying one or more eosinophil-specific antigen profiles that yielded flow cytometric data that was statistically consistent with direct counts. We found that anti-CD193 (CCR3) and anti-CD125 (IL-5Rα) antibodies were effective at detecting eosinophils in bone marrow of interleukin-5 transgenic mice, but these antibodies under-reported the percent positive cells. In contrast, anti-Siglec F alone or in combination with anti-CD45 can be used for the quantitative detection of eosinophils in mouse bone marrow and spleen. The antigen profile CD45(+)SiglecF(+)CD11c(-) was effective at detecting eosinophils in the lung as well as bone marrow and spleen, and the results obtained correlated with direct morphometric counts under all conditions evaluated (r(2)=0.98-0.99). To the best of our knowledge, this is the first systematic analysis presenting definitive correlations between percent eosinophils detected by cell surface markers and direct counting of stained cells in multiple tissues and at varying degrees of eosinophilia.

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Available from: Kristin E Killoran, May 02, 2015
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    • "Numbers of positive cells were quantified by flow cytometry (FACSCanto flow cytometer, BD Biosciences, San Jose, CA). Eosinophils were identified as SiglecF+Gr-1+CD11b+CD11c- [21], [38]. Data were collected on a FACSCanto flow cytometer and analysed with FlowJo software (version 10, Tree Star, Inc). "
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    • "Eosinophilia is, therefore, an excellent marker for monitoring allergic inflammation. It was recently shown that anti-Siglec-F alone or in combination with anti-CD45 can be used for the quantitative detection of eosinophils in mouse bone marrow and spleen and that the antigen profile CD45(+)SiglecF(+)CD11c(−) was the most effective at detecting eosinophils in the lung and correlated with direct morphometric counts under all conditions evaluated [16]. "
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    ABSTRACT: Molecular imaging of lung diseases, including asthma, is limited and either invasive or non-specific. Central to the inflammatory process in asthma is the recruitment of eosinophils to the airways, which release proteases and proinflammatory factors and contribute to airway remodeling. The aim of this study was to establish a new approach to non-invasively assess lung eosinophilia during the course of experimental asthma by combining non-invasive near-infrared fluorescence (NIRF) imaging with the specific detection of Siglec-F, a lectin found predominantly on eosinophils. An ovalbumin (OVA)-based model was used to induce asthma-like experimental allergic airway disease (EAAD) in BALB/c mice. By means of a NIRF imager, we demonstrate that 48 h-72 h after intravenous (i.v.) application of a NIRF-labeled anti-Siglec-F antibody, mice with EAAD exhibited up to 2 times higher fluorescence intensities compared to lungs of control mice. Furthermore, average lung intensities of dexamethasone-treated as well as beta-escin-treated mice were 1.8 and 2 times lower than those of untreated, EAAD mice, respectively and correlated with the reduction of cell infiltration in the lung. Average fluorescence intensities measured in explanted lungs confirmed the in vivo findings of significantly higher values in inflamed lungs as compared to controls. Fluorescence microscopy of lung cryosections localized the i.v. applied NIRF-labeled anti-Siglec-F antibody predominantly to eosinophils in the peribronchial areas of EAAD lungs as opposed to control lungs. We show that monitoring the occurrence of eosinophils, a prominent feature of allergic asthma, by means of a NIRF-labeled antibody directed against Siglec-F is a novel and powerful non-invasive optical imaging approach to assess EAAD and therapeutic response in mice over time.
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